Phospho-HP1γ (Ser83) Antibody

2600:

Immunofluorescence , Immunoprecipitation , Western Blotting

Phospho-HP1γ (Ser83) Antibody detects endogenous levels of HP1γ protein only when phosphorylated on Ser83 (also referred to as Ser93 of the unprocessed form of HP1γ). This antibody does not cross-react with HP1α or HP1β proteins.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to amino acids surrounding Ser83 of human HP1γ. Antibodies are purified by affinity chromatography.

Western Blotting

Western blot analysis of whole cell extracts from HeLa cells, untreated (lanes 1 and 4), treated for 1 h with Forskolin (30 μM) and IBMX (0.5 mM) (lanes 2 and 3), or treated for 16 h with paclitaxel (500 nM) (lanes 5 and 6), using Phospho-HP1γ (Ser83) Antibody (upper panel) or HP1γ Antibody #2619 (lower panel).

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left), forskolin- and IBMX-treated (center) or λ phosphatase-treated (right), using Phospho-HP1γ (Ser83) Antibody (green). Actin filaments have been labeled with Alexa Fluor^® 555 phalloidin (red).

Heterochromatin protein 1 (HP1) is a family of heterochromatic adaptor molecules involved in both gene silencing and higher order chromatin structure (1). All three HP1 family members (α, β, and γ) are primarily associated with centromeric heterochromatin; however, HP1β and γ also localize to euchromatic sites in the genome (2,3). HP1 proteins are approximately 25 kDa in size and contain a conserved amino-terminal chromodomain, followed by a variable hinge region and a conserved carboxy-terminal chromoshadow domain. The chromodomain facilitates binding to histone H3 tri-methylated at Lys9, a histone "mark" closely associated with centromeric heterochromatin (4,5). The variable hinge region binds both RNA and DNA in a sequence-independent manner (6). The chromoshadow domain mediates the dimerization of HP1 proteins, in addition to binding multiple proteins implicated in gene silencing and heterochromatin formation, including the SUV39H histone methyltransferase, the DNMT1 and DNMT3a DNA methyltransferases, and the p150 subunit of chromatin-assembly factor-1 (CAF1) (7-9). In addition to contributing to heterochromatin formation and propagation, HP1 and SUV39H are also found complexed with retinoblastoma (Rb) and E2F6 proteins, both of which function to repress euchromatic gene transcription in quiescent cells (10,11). HP1 proteins are subject to multiple types of post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, and sumoylation, suggesting multiple means of regulation (12-14).

HP1γ is phosphorylated on Ser83 by protein kinase A (PKA) in vitro , and activation of PKA by forskolin and IBMX treatment leads to increased phosphorylation in vivo (14). Phosphorylation of HP1γ on Ser83 also increases during mitosis as demonstrated by the Phospho-HP1γ (Ser83) Antibody, which shows increased immunofluorescent staining in untreated mitotic cells and increased Western blot signal in lysates from cells arrested in mitosis by treatment with paclitaxel. Phosphorylation of Ser83 only occurs on a subpopulation of HP1γ found associated with euchromatin, specifically HP1γ bound to coding regions of active genes (14). ?This phosphorylation impairs the ability of HP1γ to silence transcription and may be a marker for transcription elongation (14).

1. Maison, C. and Almouzni, G. (2004) Nat. Rev. Mol. Cell Biol. 5, 296-304.
2. Minc, E. et al. (2000) Cytogenet. Cell Genet. 90, 279-284.
3. Nielsen, A.L. et al. (2001) Mol. Cell 7, 729-739.
4. Lachner, M. et al. (2001) Nature 410, 116-120.
5. Bannister, A.J. et al. (2001) Nature 410, 120-124.
6. Muchardt, C. et al. (2002) EMBO Rep. 3, 975-981.
7. Yamamoto, K. and Sonoda, M. (2003) Biochem. Biophys. Res. Commun. 301, 287-292.
8. Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-2312.
9. Murzina, N. et al. (1999) Mol. Cell 4, 529-540.
10. Nielsen, S.J. et al. (2001) Nature 412, 561-565.
11. Ogawa, H. et al. (2002) Science 296, 1132-1136.
12. Minc, E. et al. (1999) Chromosoma 108, 220-234.
13. Zhao, T. et al. (2001) J. Biol. Chem. 276, 9512-9518.
14. Lomberk, G. et al. (2006) Nat. Cell Biol. 8, 407-415.

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For Research Use Only. Not For Use In Diagnostic Procedures.